LOCUS       sequence-348283-        5489 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pCAG-puro-T2A-TetON3G-polyA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5489)
  AUTHORS   Hsu CL, Lee EX, Gordon KL, Paz EA, Shen WC, Ohnishi K, Meisenhelder 
            J, Hunter T, La Spada AR
  TITLE     MAP4K3 mediates amino acid-dependent regulation of autophagy via 
            phosphorylation of TFEB.
  JOURNAL   Nat Commun. 2018 Mar 5;9(1):942. doi: 10.1038/s41467-018-03340-7.
  PUBMED    29507340
REFERENCE   2  (bases 1 to 5489)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2018-03-5"; volume: "9(1):942. doi"; pages: " 
            10.1038/s41467-018-03340-7"
COMMENT     Sequence Label: 3919-32
FEATURES             Location/Qualifiers
     source          1..5489
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          160..1176
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1100..1122
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1184..1203
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1237..1833
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGSRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1237..1256)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1733..1753
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             1852..1905
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             1906..2652
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     polyA_signal    2663..3285
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2862..2881)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     rep_origin      complement(3434..4022)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3514..3533)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        4242..4344
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4345..5004
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4411..4430)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     enhancer        5068..5371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggggcgcgc cmggsggggg gggggggggs grggggsggg gsggggsgrg gcggagaggt
       61 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
      121 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
      181 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
      241 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg
      301 gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag
      361 ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc
      421 gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt
      481 tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg
      541 gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg
      601 ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg agttgctgag
      661 cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct cgccgtgccg
      721 ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg ggccggggrg
      781 ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
      841 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
      901 atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg
      961 aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc
     1021 cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg
     1081 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
     1141 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
     1201 gtgctgtctc atcattttgg caaagaattc gccaccatga ccgagtacaa gcccacggtg
     1261 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     1321 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     1381 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     1441 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     1501 gccgagatcg gctcgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     1561 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     1621 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     1681 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     1741 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     1801 cgcacctggt gcatgacccg caagcccggt gccctgtaca agcccgggcg cgagggcaga
     1861 ggaagtcttc taacatgcgg tgacgtggag gagaatcccg gccctatgtc tagactggac
     1921 aagagcaaag tcataaactc tgctctggaa ttactcaatg gagtcggtat cgaaggcctg
     1981 acgacaagga aactcgctca aaagctggga gttgagcagc ctaccctgta ctggcacgtg
     2041 aagaacaagc gggccctgct cgatgccctg ccaatcgaga tgctggacag gcatcatacc
     2101 cactcctgcc ccctggaagg cgagtcatgg caagactttc tgcggaacaa cgccaagtca
     2161 taccgctgtg ctctcctctc acatcgcgac ggggctaaag tgcatctcgg cacccgccca
     2221 acagagaaac agtacgaaac cctggaaaat cagctcgcgt tcctgtgtca gcaaggcttc
     2281 tccctggaga acgcactgta cgctctgtcc gccgtgggcc actttacact gggctgcgta
     2341 ttggaggaac aggagcatca agtagcaaaa gaggaaagag agacacctac caccgattct
     2401 atgcccccac ttctgaaaca agcaattgag ctgttcgacc ggcagggagc cgaacctgcc
     2461 ttccttttcg gcctggaact aatcatatgt ggcctggaga aacagctaaa gtgcgaaagc
     2521 ggcgggccga ccgacgccct tgacgatttt gacttagaca tgctcccagc cgatgccctt
     2581 gacgactttg accttgatat gctgcctgct gacgctcttg acgattttga ccttgacatg
     2641 ctccccggct aatgactcga gcgggtggca tccctgtgac ccctccccag tgcctctcct
     2701 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     2761 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     2821 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     2881 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     2941 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     3001 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     3061 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     3121 tgctcccttc cctgtccttc tgattttaaa ataactatac cagcaggagg acgtccagac
     3181 acagcatagg ctacctggcc atgcccaacc ggtgggacat ttgagttgct tgcttggcac
     3241 tgtcctctca tgcgttgggt ccactcagta gatgcctgtt gaattgggta cgcggccagc
     3301 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     3361 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     3421 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3481 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3541 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3601 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3661 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3721 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3781 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3841 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3901 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3961 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4021 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4081 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttcgaccgaa
     4141 taaatacctg tgacggaaga tcacttcgca gaataaataa atcctggtgt ccctgttgat
     4201 accgggaagc cctgggccaa cttttggcga aaatgagacg ttgatcggca cgtaagaggt
     4261 tccaactttc accataatga aataagatca ctaccgggcg tattttttga gttgtcgaga
     4321 ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac caccgttgat
     4381 atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc tcaatgtacc
     4441 tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa gaaaaataag
     4501 cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc tcatccggaa
     4561 ttacgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca cccttgttac
     4621 accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata ccacgacgat
     4681 ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttatggtga aaacctggcc
     4741 tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc ctgggtgagt
     4801 ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc cgttttcacc
     4861 atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat tcaggttcat
     4921 catgccgttt gtgatggctt ccatgtcggc agaatgctta atgaattaca acagtactgc
     4981 gatgagtggc agggcggggc gtaatactag ttattaatag taatcaatta cggggtcatt
     5041 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     5101 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     5161 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     5221 ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa
     5281 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     5341 catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt tctgcttcac
     5401 tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt tttaattatt
     5461 ttgtgcagcg atgggggcgg ggggggggg
//
